Synthesis, DFT and antimicrobial activity assays in vitro for novel cis/trans-but-2-enedioic acid esters

Article abstract of DOI:10.1016/j.molstruc.2014.01.063

Six novel cis/trans-but-2-enedioic acid esters had been synthesized to discover the new bioactive molecules that could kill food-related bacteria and fungi. Their structures were analyzed by melting point, LC-MS, ¹H NMR and ¹³C NMR. 4-(Methoxycarbonyl) phenyl ethyl fumarate (6b) was also characterized by single-crystal X-ray diffraction. Their antimicrobial activities were evaluated in vitro by measuring the minimal inhibitory concentration (MIC). Compared with the single monomethyl fumarate and methyl 4-hydroxybenzoate, these compounds had stronger antimicrobial activity against all the eight microorganisms. Among the antibacterial and antifungal compounds, 4-(methoxycarbonyl) phenyl methyl fumarate (6a) showed the best antimicrobial activity. The electronic properties of these compounds were calculated by the density functional theory (DFT) method with 6-31G (d, p) basis set. DFT studies indicated that molecular electrostatic potential (MEP) map, E_LUMO, energy gap, electronegativity and electrophilicity index could be helpful to understand the various antimicrobial activities among these compounds. The antimicrobial activity of compound 6a was evaluated in vitro against Salmonella choleraesuis subsp. choleraesuis, Lactococcus lactis subsp. lactis and Saccharomyces cerevisiae by time-kill, and it was found that compound 6a exhibited significant microbiocidal activity against the three microorganisms.

Full text of DOI:10.1016/j.molstruc.2014.01.063

Accepted Manuscript
Synthesis, DFT and antimicrobial activity assays in vitro for novel cis/trans-
but-2-enedioic acid esters
Yan-Long Ma, Ru-Jin Zhou, Xing-Ye Zeng, Ya-Xiong An, Song-Shan Qiu, Li-
Jun Nie
PII:
S0022-2860(14)00124-0
DOI:
http://dx.doi.org/10.1016/j.molstruc.2014.01.063
MOLSTR 20340
Reference:
Journal of Molecular Structure
To appear in:
Received Date:
Revised Date:
Accepted Date:
12 November 2013
19 January 2014
27 January 2014
Please cite this article as: Y-L. Ma, R-J. Zhou, X-Y. Zeng, Y-X. An, S-S. Qiu, L-J. Nie, Synthesis, DFT and
antimicrobial activity assays in vitro for novel cis/trans-but-2-enedioic acid esters, Journal of Molecular
Structure (2014), doi: http://dx.doi.org/10.1016/j.molstruc.2014.01.063
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Synthesis, DFT and antimicrobial activity assays in vitro for novel
cis/trans-but-2-enedioic acid esters
Yan-Long Maa,b, Ru-Jin Zhoua,b, *, Xing-Ye Zengb, Ya-Xiong Ana, Song-Shan Qiub,
Li-Jun Nieb
a
School of biological and chemical engineering, Jiangsu University of Science and
Technology, Zhenjiang 212003, China
b
Department of Petrochemical Engineering, Guangdong University of Petrochemical
Technology, Maoming 525000, China
* Corresponding author. Tel.: +86 668 2923672; fax: +86 668 2923735; E-mail
address: rujinzhou@126.com (R.-J. Zhou).
Highlights
ꢀ
ꢀ
Six novel cis/trans-but-2-enedioic acid esters were synthesized and characterized.
Compound 6a showed the strongest antimicrobial activity against all tested
microorganisms.
ꢀ
SAR studies showed that these compounds might possess electrophilic reactivity
towards biological nucleophiles.
Abstract
Six novel cis/trans-but-2-enedioic acid esters had been synthesized to discover the
new bioactive molecules that could kill food-related bacteria and fungi. Their
1
structures were analyzed by melting point, LC-MS, H NMR and 13C NMR.
4-(methoxycarbonyl) phenyl ethyl fumarate (6b) was also characterized by
single-crystal X-ray diffraction. Their antimicrobial activities were evaluated in vitro
by measuring the minimal inhibitory concentration (MIC). Compared with the single
1

monomethyl fumarate and methyl 4-hydroxybenzoate, these compounds had stronger
antimicrobial activity against all the eight microorganisms. Among the antibacterial
and antifungal compounds, 4-(methoxycarbonyl) phenyl methyl fumarate (6a) showed
the best antimicrobial activity. The electronic properties of these compounds were
calculated by the density functional theory (DFT) method with 6-31G (d, p) basis set.
DFT studies indicated that molecular electrostatic potential (MEP) map, ELUMO
,
energy gap, electronegativity and electrophilicitu index could be helpful to understand
the various antimicrobial activities among these compounds. The antimicrobial
activity of compound 6a was evaluated in vitro against S. choleraesuis subsp.
choleraesuis, L. lactis subsp. lactis and S. cerevisiae by time-kill, and it was found
that compound 6a exhibited significant microbiocidal activity against the three
microorganisms.
Keywords: Synthesis; Cis/trans-but-2-enedioic acid esters; Antimicrobial activity
assay; DFT studies; Time-kill assay
1. Introduction
Fumarate esters and hydroxybenzoic acid esters have played an important role in
the field of food and pharmaceutical industries owing to their low cost, low toxicity
and a broad spectrum of activity against molds, yeasts and bacteria [1-5]. Since the
biological activity of fumarate esters and hydroxybenzoic acid esters is discovered,
their derivatives have already been prepared and considered as potential food
additives and therapeutic compounds, although only a few of them have been used as
food preservatives and clinical medicine. For example, dimethyl fumarate,
monomethyl fumarate and monoethyl fumarate are widely used as food preservatives
and oral therapeutic agent for the treatment of psoriasis and multiple sclerosis [6, 7];
Methyl 4-hydroxybenzoate is considered to be a relatively safe compound with low
potential bioaccumulation and is still used as preservative in the food and cosmetic
industries [8, 9]; Methyl 2-hydroxybenzoate (methyl salicylate) is an active ingredient
in many topical analgesic preparations for ameliorating painful musculoskeletal
2

disorders of various aetiologies [10].
However, the resistance of pathogens toward currently available drug therapy is
rapidly becoming a major worldwide problem. Thus, the development of new
compounds resistant for fungi and bacteria has become one of the most important
areas of antibacterial research. Fungi and bacteria are extremely difficult to control in
food industry and also responsible for the great economic losses every year. In
addition, traditional food preservatives are not effective and cause negative impact on
human health [11, 12]. Therefore, researching new antifungal and antibacterial agents
remains an urgent task in food science. In this paper, the active structures of
cis/trans-but-2-enedioic acid monoesters and hydroxybenzoic acid methyl ester were
combined to synthesize novel cis/trans-but-2-enedioic acid esters to discover new
bioactive molecules as potential food preservatives against fungi and bacteria (see
1
Scheme 1). All the compounds were analyzed by LC-MS, H NMR and 13C NMR,
and the compound 6b were also characterized by X-ray crystallography.
Quantitative structure-activity relationships (QSAR) have been used to correlate
biological activity with some appropriate quantum chemical descriptors [13, 14] such
as EHOMO, ELUMO, energy gap, dipole moment, NBO charge, molecular electrostatic
potential (MEP) map, etc. Recently, there is enough evidence that quantum chemical
descriptors based on DFT calculation often provide much desirable information in
QSAR studies [15-17]. The present study was aimed to report the synthesis of the
novel compounds and to investigate their antimicrobial activity on the basis of in vitro
and DFT calculations.
2. Materials and Methods
2.1. Reagents and equipments
All reagents and reactants were obtained from commercial suppliers and were of
analytical grade. Melting points were determined by a DCS-500 binocular microscope
(Shanghai Innuo Precision Instrument Co., Shanghai, China). The purification of
synthesized compounds was achieved by passage through column chromatography on
silica gel (200-300 mesh, Qingdao Haiyang Chemical Co., Qingdao, China).
Analytical thin layer chromatography (TLC) was performed on silica gel GF254. The
3

purity of all the compounds was determined by HPLC of SHIMADZU (SPD-20A
ultraviolet absorption, LC-20AT volumetric infusion pump). Samples were evaluated
under the 254 nm condition in a 80:20 MeOH: H2O solution containing 0.1% formic
acid (v/v). Mass spectra (MS) were recorded on a SHIMADZU LC/MS-2020.
Samples were evaluated in the positive-negative mode in a 1:1 CH3CN: H2O solution
1
with addition of 0.1% formic acid (v/v). H (400 MHz) and 13C (101 MHz) NMR
spectra were recorded on Bruker AVANCEⅡ400 spectrometer with Me4Si as the
internal standard. Chemical shifts (δ values) and coupling constants (J values) were
given in ppm and Hz, respectively. But the solvent signal and water signal were also
measured (CDCl3: δ =7.27; DMSO-d6: δ =2.51, δ =39.5, δ =3.34 for H2O). The
H
H
C
H
anhydrous tetrahydrofuran (THF) solvent was dried and purified according to
standard techniques.
2.2. Synthesis
General procedure for the synthesis of cis-but-2-enedioic acid monoesters 1a-c
A mixture of anhydrous Na2CO3 (5.513 g, 0.052 mol) in corresponding monohydric
alcohol (40-50 mL) were slowly added maleic anhydride (10 g, 0.102 mol). The
mixture was stirred at room temperature for 0.5-1 h. The solvent was removed under
reduced pressure. The residue was dissolved in 20 mL of water and then the mixture
was acidified with 9 mL of concentrated hydrochloric acid. The aqueous phase was
extracted with ethyl acetate (3×40 mL). The organic extracts were collected, washed
with brine (2×20 mL), dried over anhydrous Na2SO4, filtrated and evaporated to give
the pure cis-but-2-enedioic acid monoesters.
1
Monomethyl maleate (1a) Colorless oil; Yield 12.7 g (95.7%); HPLC: 94.3%; H
NMR (400 MHz, CDCl3): δ 6.43 (d, J = 12.5 Hz, 1H), 6.38 (d, J = 12.5 Hz, 1H), 3.88
(s, 3H); MS (ESI) m/z: 114.9 [M-CH3] ¯, 174.9 [M+HCOO] ¯, 230.9 [2M-H] ¯.
1
Monoethyl maleate (1b) Colorless oil; Yield 14.31 g (97.3%); HPLC: 96.8%; H
NMR (400 MHz, CDCl3): δ 6.43 (d, J = 12.6 Hz, 1H), 6.37 (d, J = 12.6 Hz, 1H), 4.34
(q, J = 7.1 Hz, 2H), 1.36 (t, J = 7.2 Hz, 3H); MS (ESI) m/z: 114.9 [M-CH2CH3]¯,
142.9 [M-H]¯, 188.9 [M+HCOO]¯, 287.0 [2M-H]¯.
4

1
Monopropyl maleate (1c) Colorless oil; Yield 15.6 g (98.6%); HPLC: 97.5%; H
NMR (400 MHz, CDCl3): δ 6.43 (d, J = 12.6 Hz, 1H), 6.38 (d, J = 12.6 Hz, 1H), 4.23
(t, J = 6.7 Hz, 2H), 1.81 – 1.69 (m, 2H), 0.99 (t, J = 7.4 Hz, 3H); MS (ESI) m/z: 114.9
[M-CH2CH2CH3]¯, 156.9 [M-H]¯, 202.9 [M+HCOO]¯, 315.0 [2M-H]¯.
General procedure for the synthesis of trans-but-2-enedioic acid monoesters 2a-c
Anhydrous AlCl3 (0.16 g) was added to the cis-but-2-enedioic acid monoesters (4 g)
under stirring. The mixture was heated at 60-70 ℃ for 0.5-2 h. The cooled mixture
was diluted with 1 N HCl (30 mL) and the aqueous phase extracted with ethyl acetate
(3×40 mL). The organic extracts were collected, washed with brine (2×20 mL), dried
over anhydrous Na2SO4, filtrated and evaporated to give crude trans-but-2-enedioic
acid monoesters.
Monomethyl fumarate (2a) White crystals after crystallization with water; Yield
1
3.3 g (82.5%); HPLC: 92.0%; H NMR (400 MHz, CDCl3): δ 6.95 (d, J = 15.8 Hz,
1H), 6.87 (d, J = 15.8 Hz, 1H), 3.84 (s, 3H); MS (ESI) m/z: 129.0 [M-H] ¯, 174.9
[M+HCOO]¯, 258.9.0 [2M-H]¯.
Monoethyl fumarate (2b) White crystals after crystallization with ether-petroleum
ether; Yield 2.32 g (58%); HPLC: 98.6%; 1H NMR (400 MHz, CDCl3): δ 6.95 (d, J =
15.8 Hz, 1H), 6.85 (d, J = 15.8 Hz, 1H), 4.28 (q, J = 7.1 Hz, 2H), 1.34 (t, J = 7.1 Hz,
3H); MS (ESI) m/z: 142.9 [M-H]¯, 188.9 [M+HCOO]¯, 287.0 [2M-H]¯.
Monopropyl fumarate (2c) White crystals after crystallization with diethyl
1
ether-petroleum ether; Yield 1.42 g (35.5%); HPLC: 98.6%; H NMR (400 MHz,
CDCl3): δ 6.95 (d, J = 15.8 Hz, 1H), 6.85 (d, J = 15.8 Hz, 1H), 4.18 (t, J = 6.7 Hz,
2H), 1.78 – 1.67 (m, 2H), 0.98 (t, J = 7.4 Hz, 3H); MS (ESI) m/z: 157.0 [M-H]¯, 203.0
[M+HCOO]¯, 315.0 [2M-H]¯.
Synthesis of 2-(methoxycarbonyl) phenyl methyl maleate (3)
Oxalyl chloride (1.7 g, 0.013 mol) was added dropwise to a solution of
monomethyl maleate (1.1 g, 0.0085 mol) and two drops of DMF in 20 mL of
anhydrous THF at 0 ℃. Then the resultant mixture was stirred at room temperature
for 2 h. After completion of the reaction, the solvent was evaporated. (Z)-methyl
5

4-chloro-4-oxobut-2-enoate (3a) was obtained. To a stirred solution of methyl
2-hydroxybenzoate (1 g, 0.0066 mol) and NaH (0.32 g, 0.013 mol) in anhydrous THF
(15 mL) was added compound 3a at 0 ℃. The mixture was stirred at room
temperature for 1-2 h. After completion of the reaction, the pH of the mixture was
adjusted to 4 with 1 N HCl. The solvent was evaporated and the aqueous was
extracted with ethyl acetate (3×20 mL). The organic extracts were collected, washed
with brine (2×20 mL), dried over anhydrous Na2SO4, filtrated and evaporated to give
a colorless oil. The crude material was purified by chromatography on silica gel with
ethyl acetate-petroleum ether (10:1) to give 1.2 g of a solid (yield 69%). mp:
45-46 ℃; 1H NMR (400 MHz, DMSO-d6): δ 7.98 (dd, J = 7.8, 1.6 Hz, 1H), 7.73 (td,
J = 7.9, 1.7 Hz, 1H), 7.46 (td, J = 7.7, 1.1 Hz, 1H), 7.35 (dd, J = 8.1, 0.8 Hz, 1H), 7.01
(d, J = 0.5 Hz, 2H), 3.79 (s, 3H), 3.76 (s, 3H); 13C NMR (101 MHz, DMSO-d6): δ
164.90, 164.40, 163.27, 149.62, 134.88, 134.72, 132.41, 131.54, 127.04, 124.08,
122.84, 52.67, 52.53; MS (ESI) m/z: 248.8 [M-CH3]¯.
Similarly, the rest of compounds were synthesized by the same method.
2-(methoxycarbonyl) phenyl methyl fumarate (4) White solid; Yield 1.3 g
(74.7%); mp: 47-48 ℃; HPLC: 97.9%; 1H NMR (400 MHz, DMSO-d6): δ 7.99 (dd, J
= 7.8, 1.6 Hz, 1H), 7.74 (td, J = 7.9, 1.7 Hz, 1H), 7.47 (td, J = 7.7, 1.0 Hz, 1H), 7.36
(dd, J = 8.1, 0.8 Hz, 1H), 7.01 (s, 2H), 3.80 (s, 3H), 3.77 (s, 3H). 13C NMR (101 MHz,
DMSO-d6): δ 164.66, 164.16, 163.03, 149.39, 134.64, 134.47, 132.18, 131.30, 126.79,
123.84, 122.61, 52.43, 52.28; MS (ESI) m/z: 248.8 [M-CH3] ¯.
3-(methoxycarbonyl) phenyl methyl fumarate (5) White solid; Yield 1.26 g
1
(72.4%); mp: 75-76 ℃; HPLC: 96.0%; H NMR (400 MHz, DMSO-d6): δ 7.93 –
7.87 (m, 1H), 7.84 – 7.79 (m, 1H), 7.63 (t, J = 7.9 Hz, 1H), 7.55 (ddd, J = 8.1, 2.2, 1.0
13
Hz, 1H), 7.00 (s, 2H), 3.87 (s, 3H), 3.80 (s, 3H); C NMR (101 MHz, DMSO-d6): δ
165.33, 164.67, 162.92, 150.07, 134.41, 132.40, 131.09, 130.17, 126.94, 126.59,
122.31, 52.41, 52.37; MS (ESI) m/z: 248.8 [M-CH3]¯.
4-(methoxycarbonyl) phenyl methyl fumarate (6a) White solid; Yield 1.5 g
6

1
(86.2%); mp: 111-112 ℃; HPLC: 96.6%; H NMR (400 MHz, DMSO-d6): δ 8.08 –
8.05 (m, 1H), 8.05 – 8.03 (m, 1H), 7.43 – 7.41 (m, 1H), 7.41 – 7.38 (m, 1H), 7.01 (s,
2H), 3.87 (s, 3H), 3.79 (s, 3H); 13C NMR (101 MHz, DMSO-d6): δ 165.45, 164.63,
162.61, 153.66, 134.63, 132.28, 130.84, 127.57, 122.08, 52.44, 52.25; MS (ESI) m/z:
248.8 [M-CH3]¯.
4-(methoxycarbonyl) phenyl ethyl fumarate (6b) White powder; Yield 1.42 g
1
(77.6%); mp: 69-70 ℃; HPLC: 97.3%; H NMR (400 MHz, DMSO-d6): δ 8.07 –
8.05 (m, 1H), 8.05 – 8.03 (m, 1H), 7.43 – 7.41 (m, 1H), 7.41 – 7.38 (m, 1H), 6.99 (s,
2H), 4.25 (q, J = 7.1 Hz, 2H), 3.87 (s, 3H), 1.28 (t, J = 7.1 Hz, 3H); 13C NMR (101
MHz, DMSO-d6): δ 165.44, 164.10, 162.60, 153.66, 134.95, 132.12, 130.82, 127.55,
122.06, 61.26, 52.22, 13.89; MS (ESI) m/z: 248.8 [M-CH2CH3]¯.
4-(methoxycarbonyl) phenyl propyl fumarate (6c) White powder; Yield 1.5 g
1
(78.1%); mp: 59-60 ℃; HPLC: 98.2%; H NMR (400 MHz, DMSO-d6): δ 8.07 –
8.05 (m, 1H), 8.05 – 8.02 (m, 1H), 7.43 – 7.41 (m, 1H), 7.41 – 7.38 (m, 1H), 7.00 (s,
2H), 4.17 (t, J = 6.6 Hz, 2H), 3.87 (s, 3H), 1.73 – 1.59 (m, 2H), 0.94 (t, J = 7.4 Hz,
3H); 13C NMR (101 MHz, DMSO-d6): δ 165.44, 164.18, 162.61, 153.67, 134.92,
132.13, 130.82, 127.55, 122.06, 66.61, 52.22, 21.37, 10.14; MS (ESI) m/z: 248.8
[M-CH2CH2CH3]¯.
2.3. Crystal structure determination
The X-ray diffraction data for compound 6b were collected by a Bruker SMART
APEX Ⅱ CCD X-ray crystallography system which was equipped with
graphite-monochromated Mo Kα radiation (λ=0.71073Å) by using ψ and ω scan
model at room temperature. The data were refined and reduced by the SMRT [18] and
SAINT [19] software, respectively. The structure was solved by direct method and
refined by a full-matrix least-squares procedure based on F2 using SHELXS-97 and
SHELXL-97 [18]. All non-hydrogen atoms were refined anisotropically and all
hydrogen atoms were generated geometrically.
2.4. Computational details
7

All calculations were performed by Gaussian 09 software package [20]. The gas
phase geometry of compound 6b in the ground state was fully optimized by density
functional theory (DFT/B3LYP) method [21-25] with 6-31G (d, p) basis set. The
crystallographic structure of compound 6b was used as the initial structure in
optimization without any symmetry restriction. In addition, the molecular structures
of other compounds (3-6a and 6c) in the ground state were also optimized by
B3LYP/6-31G (d, p). This method has been previously used for small molecule’s
calculation [26, 27]. The vibrational analysis using the analytical gradient algorithm
was performed, and the absence of imaginary frequencies confirmed that the
optimized structures were at minimum energy.
The energies, Ecation and Eanion, of the ionic states of compounds (3-6c) were
calculated at the B3LYP/6-31G (d, p) level by using the neutral geometry. Hence, the
ionization potential (IP) = Ecation-En and the electron affinity (EA) = En- Eanion values
were the vertical ionization potential and vertical electron affinity [28, 29],
respectively. From these calculations the other parameters such as electronegativity
(χ), hardness (η), softness (σ), and electrophilicity index (ω) were calculated as
2
follows: χ = (IP+EA)/2, η = (IP-EA)/2, σ = 1/η and ω = χ /2η. The mean polarizability
[30] was obtained from the diagonal elements of the polarizability tensor
1
α =
(
αxx +αyy +αzz
)
as:
3
2.5. Biological assays
In vitro antifungal and antibacterial activities’ evaluation by MIC assay. The
antimicrobial activities of the newly synthesized compounds were evaluated by the
minimum inhibitory concentration (MIC) and were tested in vitro against Gram
negative bacteria (S. choleraesuis subsp. choleraesuis ATCC 13312), Gram positive
bacteria (B. subtilis ATCC 6633 and L. lactis subsp. lactis ATCC 11454), Yeasts (C.
albicans ATCC 10231 and S. cerevisiae ATCC 9763 ) and Molds (A. flavus CICC
40191, A. niger CICC 2273 and P. chrysogenum CICC 4017) according to CLSI
reference methods including M7-A7 for bacteria [31], M27-A2 for yeasts [32] and
M38-A2 for molds [33], respectively. The results were compared with that of standard
8

compounds (monomethyl fumarate and methyl 4-hydroxybenzoate). Briefly, bacteria
were cultured in nutrient broth (NB) at 37 ℃ for 24 h while B. subtilis was cultured
at 30 ℃. Yeasts were grown on Sabouraud dextrose broth (SDB) at 30 ℃ for 24 h.
Molds were grown on sabouraud dextrose agar (SDA) slants at 28 ℃ for 7 days and
the spores were collected by sterile saline (0.85%). The cell suspension was diluted to
achieve the turbidity of 0.5 McFarland turbidity standard (~108 for bacteria and 106
for fungi), which was used to inoculate in NB for bacteria and SDB for fungi,
respectively. Suspensions were further diluted 1:50 with the corresponding medium to
obtain a final inoculum of 104 CFU/mL. The newly synthesized compounds and
standard compounds were prepared by double dilution method in DMSO, and the
final concentration of the newly synthesized compounds and standard compounds in
the tested medium was 18.75, 25, 37.5, 50, 75, 100, 150, 200 µg/mL and 100, 200,
300, 400, 600, 800 µg/mL, respectively. Bacteria were cultured in NB at 37 ℃ for
24 h except for B. subtilis at 30 ℃, yeasts were cultured in SDB at 30 ℃ for 24 h
and molds were cultured in SDB at 28 ℃ for 48 h. The concentration of DMSO in
the tested medium did not exceed 2% (v/v) of the total solution composition. The MIC
was defined as the lowest concentration of the tested compounds without visible
microorganism growth after incubation. All determinations were performed in
triplicate.
Time-kill assays. The antibacterial activity against S. choleraesuis subsp.
choleraesuis ATCC 13312 and L. lactis subsp. lactis ATCC 11454 and antifungal
activity against S. cerevisiae ATCC 9763 of compound 6a were further analyzed by
time-kill. Bacteria and yeasts were diluted to the final concentration (~104 CFU/mL).
The compound 6a was added into the medium to produce final concentration
equivalents to 3/4×MIC, 1×MIC and 1.5×MIC value, respectively. Control
experiment was conducted without compound 6a. The culture conditions were done
the same as above MIC study. At predetermined time points (0, 3, 6, 9 and 24 h), a
100 µL suspension was diluted using 0.85% saline solution by ten times dilution
9

method. Then, 100 µL samples were coated on nutrient agar or SDA plates and
incubated for 24 h. The colonies were counted within 30 and 300 counts for each
dilution. All of the experiments were performed in triplicate.
3. Results and Discussion
3.1. Compound characterizations
All compounds (1a-6c) were synthesized according to Scheme 1. The monomethyl
maleate and fumaric acid monoester were prepared in this work and these compounds
were used as the starting materials for further synthesis. The structure of these
1
compounds had been analyzed and identified by LC-MS and H NMR spectrum. In
1
the H NMR spectra of the newly synthesized compounds, peaks in the range of δ
7.35-8.08 ppm showed phenyl protons. The signals around 7.00 ppm for two protons
were confirmed to be the presence of -CH=CH- group. The other alkyl proton signals
were observed between 0.94 and 4.25 ppm. In 13C NMR, peaks around δ 162-165
ppm showed the ester carbons, peaks in the range of δ 122-153 ppm confirmed the
presence of phenyl and -CH=CH- group, and peaks in the range of around δ 10-66
ppm showed the aliphatic carbons. Compound 3 and 4, which were difficult to
distinguish each other, had extremely similar peaks at corresponding chemical shifts
1
13
and coupling constants in their H NMR and C NMR spectra but they had different
melting points and chromatographic retention times. The melting points of compound
3 and 4 were 45-46 ℃ and 47-48 ℃, and the chromatographic retention times of
compound 3 and 4 were 4.28 and 7.32 min, respectively. The molecular structure and
cell packing of compound 6b were shown in Fig. 1. The crystal data and its collection
and refinement parameters of 6b were shown in Table 1.
3.2. DFT studies
3.2.1. Geometry optimization
The calculated molecular structure of compound 6b was constructed on the basis of
its crystallographic data and the final geometry was confirmed as minima of the
potential energy surfaces with calculation of the hessians showing no imaginary
frequencies. The most significant differences between the X-ray and the calculated
10

values of the dihedral angles were at O(2)-C(2)-C(3)-C(4), C(7)-C(6)-O(3)-C(9) and
C(12)-O(6)-C(13)-C(14)(see Fig. 1 and 2). It should be note that the theoretical
calculations performed on a single molecule in the gaseous state contrary to the
experimental values, which were recorded in presence of intermolecular interactions
[27, 36]. In spite of these differences, most of the geometrical parameters of
theoretical studies were nearly the same as the X-ray experimental data for compound
6b. This method was also used to calculate the other parameters for compound 6b,
and was extended to the ground state geometrical optimization of the rest compounds.
The optimized structures of the newly synthesized compounds, selected dihedral
angles and NBO charges were shown in Fig. 2.
3.2.2. Molecular electrostatic potential
Molecular electrostatic potential (MEP) maps provide the isosurface values with
the location of negative and positive electrostatic potentials, and have been widely
used as a tool in understanding the sites for electrophilic attack and nucleophilic
reactions, as well as intermolecular interactions [34, 35]. MEP maps are also well
suitable for analyzing binding sites on the basis of the recognition of one molecule by
another, especially in drug design [14, 36]. To consider the most probable sites of the
molecules for an interaction with electrophilic and nucleophilic species, MEP was
calculated at the B3LYP-6-31G (d, p) optimized geometry. The different values of the
electrostatic potential at the surface were marked by different colors. In Fig. 2, red
represented regions of most electro negative electrostatic potential, blue represented
regions of most positive electrostatic potential and green represented regions of zero
potential. The carbonyl oxygen atoms were surrounded by a greater negative charge
surface, thus these sites were potentially more favorable for electrophilic attack and
should be possible to bind with the active drug-receptor sites.
3.2.3. Frontier molecular orbitals
The frontier molecular orbitals play an important role in several chemical and
pharmacological processes. EHOMO measures the electron donating ability of the
molecule, while ELUMO indicates the ability of molecule to accept electrons [37, 38].
Thus the bigger the EHOMO is, the greater the electron donating capability, and the
11

smaller the ELUMO is, the smaller the resistance to accept electrons. Moreover the
energy gap (ΔE) between HOMO and LUMO determines the reactivity of the
molecule. A molecule with a small ΔE is more polarizable and also generally
associated with a high chemical reactivity. The electron density of HOMO and LUMO
molecular obitals for the newly synthesized compounds were shown in Fig. 3. The
major part of electron density of HOMO was located on phenyl ring, while that of
LUMO was on ester groups of cis/trans-but-2-enedioic acid monoesters. As shown in
Table 2, the ranking of the isomers (3-6a) and homologous compounds (6a-6c)
according to their ability to accept electrons were: 6a > 5 > 4 > 3, 6a > 6b > 6c,
respectively. The calculations from Table 2 also showed that the reactivity abilities of
the isomers (3-6a) and homologous compounds (6a-6c) decreased in the following
order: 6a > 5 > 4 > 3, 6a > 6c > 6b, respectively. The energy gap and ELUMO of
compound 6a were 4.3092 and -2.5459 eV, respectively. The dipole moment (μ) of the
newly synthesized compounds were also calculated and listed in Table 2.
3.2.4. Global descriptors
The values of quantum chemical parameters, calculated for each of the newly
synthesized compounds using the vertical IPs and EAs, were given in Table 2.
Hardness is a direct measure of the stability of the molecule, and softness provides a
measure of its reactivity [39]. Electrophilicity index measures the propensity of
chemical specie to accept electrons [40, 41]. Hence, the higher the electrophilicity
index, the greater the propensity of the complex to attract electrons from a donor
molecule. A good nucleophile is characterized by low values of ω, whereas a good
electrophile is characterized by high values of ω. It was clear in Table 2 that the
molecules had high electrophilicity index values and were good electrophiles.
Electronegativity is a measure of the tendency of the molecule to attract electrons [42,
43]. Table 2 showed that the higher the value of electronegativity, the lower was the
value of ELUMO and the easier for the electrons to be easily attracted.
3.3. Antimicrobial activity and structure-activity relationship (SAR)
The antimicrobial activities of the newly synthesized compounds against S.
12

choleraesuis subsp. choleraesuis, B. subtilis, L. lactis subsp. lactis, C. albicans, S.
cerevisiae, A. flavus, A. niger and P. chrysogenum were performed. As shown in Table
3, the results revealed that these newly synthesized compounds exhibited the capacity
of inhibiting the growth of the tested microorganisms, they were more effective than
monomethyl fumarate (MMF) and methyl 4-hydroxybenzoate (MP) and most of the
newly synthesized compounds were effective against bacteria and fungi within
200µg/mL. In particular, compound 6a was the most effective derivative for
antimicrobial activity against all tested microorganisms. Until now, the QSAR studies
[44-47] shows that compounds with an α, β-unsaturated carbonyl moiety are more
reactive than simple carbonyls. Because of its conjugation with the carbonyl group,
the β-carbon is positively polarized and becomes the preferred site of nucleophilic
attack. They could react with nucleophilic groups on peptides, proteins, or DNA, thus
causing disruption of these biological macromolecules and hence cause toxic effects
[48, 49]. These compounds represent α, β-unsaturated carbonyl moiety responsible for
the toxicity of these compounds as they are expected to undergo Michael-type
addition with essential biological thiol sites [50]. Fumaric acid esters possess
electrophilic reactivity towards biological nucleophiles, and they could react with
nucleophiles such as sulfhydryl groups of biological target molecules (Scheme 2) [51].
As shown in Table 2 and 3, the antimicrobial activity of the newly synthesized
compounds was increased by increasing their electronegativity, where MIC values
correlated with lower ELUMO for these compounds. The parallelism with ELUMO
indicated that these compounds could act as electrophilic agents. The high
electrophilicity index values of these compounds also showed that they were good
electrophiles. As seen from Fig. 3, the LUMO of these compounds was mostly located
on cis/trans-but-2-enedioic acid monoesters. So these compounds might use their
cis/trans-but-2-enedioic acid monoesters fragments to accept electrons and undergo
covalently binding to amino acid side chains with nucleophilic properties, such as the
thiol group of cysteine and the amine group of lysine. The result was to change the
structure of biological macromolecules and their bioactivities.
13

Scheme 2 Reaction between DMF and thiols
On the other hand, energy gap was also a good descriptor for antimicrobial activity
for the newly synthesized compounds. Especially for the isomeric compounds 3, 4, 5
and 6a, the theoretical analysis of the energy gap (ΔE) of those isomers showed direct
correlation with their antimicrobial activity. Most of the values of the isomers’ MIC
decreased along with the decrease of the energy gap. The parallelism with the energy
gap indicated that the antimicrobial activity of these isomers could be associated with
the chemical reactivity under the same experimental conditions. However, compound
4 showed stronger antimicrobial activity against S. choleraesuis subsp. choleraesuis
more than that of compound 5, while the chemical reactivity of compound 5 was
higher than that of compound 4. This discrepancy may be associated with the cell
structure of different organisms. The MIC results indicated that the antimicrobial
activities of homologous compounds (6a-6c) were very different. It should be noted
that the lengths of alkyl chain significantly alter the biological activities. Notably, the
high hydrophilicity of these compounds could enhance their antimicrobial activity,
which was in accordance with the experimental results (see Table 3).
In summary, compound 6a had the strongest antimicrobial activity against bacteria
and fungi among all the tested compounds. In addition, a time–kill assay was
performed to further investigate the microbiocidal activity of compound 6a. The
time–kill studies were performed within 24 h with food-related microbial pathogens
being exposed to 0.75, 1 and 1.5 MIC of compound 6a. Fig. 4 showed the results of
time-kill curves for S. choleraesuis subsp. choleraesuis (A), L. lactis subsp. lactis (B)
and S. cerevisiae (C). At 0.75 and 1 MIC, compound 6a significantly decreased
microbial population within 9 h; then the pathogens began to grow. At 1.5 MIC, the
pathogens were not detected after 6 h.
4. Conclusion
In this study, six novel cis/trans-but-2-enedioic acid esters were designed and
14

synthesized. All the prepared compounds were analyzed by spectral data (1H NMR,
13C NMR and LC-MS). The structure of compound 6b was also characterized by
single-crystal X-ray diffraction. The antibacterial and antifungal activities in vitro of
these compounds against S. choleraesuis subsp. choleraesuis, B. subtilis, L. lactis
subsp. lactis, C. albicans, S. cerevisiae, A. flavus, A. niger and P. chrysogenum were
studied. The results showed that the antibacterial and antifungal activities of the newly
synthesized compounds were stronger than that of the single monomethyl fumarate
and methyl 4-hydroxybenzoate. Compound 6a showed the strongest antibacterial and
antifungal activity against all tested microorganisms (MIC≤75 µg/mL). The results of
SAR analysis indicated that the antimicrobial activity of cis/trans-but-2-enedioic acid
esters increased with decreasing values of LUMO and these compounds might possess
electrophilic reactivity towards biological nucleophiles. Furthermore, isomers (3-6a)
with higher chemical reactivity had stronger antibacterial and antifungal activity.
Homologous compounds (6a-6c) with higher hydrophilicity had stronger antibacterial
and antifungal activity. Overall, the SAR analysis provided the clues about how to
understand the various antimicrobial activities among these compounds. The
microbiocidal activity of compound 6a against S. choleraesuis subsp. choleraesuis, L.
lactis subsp. lactis and S. cerevisiae was confirmed by time–kill, and it was found that
the log values for the pathogens were significantly reduced by compound 6a at
1.5MIC.
Acknowledgments
This work was supported by the Produce Study Research Combination Project of
Guangdong Province and Ministry of Education (2009B090300134).
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Table 1. Crystal data and structure refinement of compound 6b.
CCDC no.
971415
Empirical formula
Formula weight
Temperature
C H O
14 14
6
278.25
296(2) K
Wavelength
0.71073Å
Crystal system
Space group
Triclinic
P(no.2)
Unit cell dimensions
a = 8.063(8) Å
b = 8.234(8) Å
c = 10.672(10) Å
687.5(12) Å3
2
α = 86.470(14) °
β = 85.849(14) °
γ = 76.897(14) °
Volume
Z
Density (calculated)
Absorption coefficient
F(000)
1.344 mg/m3
0.106 mm-1
292
θ range for data collection
Limiting indices
1.92-26°
-9 ≤ h ≤ 9
-10 ≤ k ≤ 10
-11 ≤ l ≤ 13
5350
Reflections collected
Independent reflections
Completeness to θ = 26°
Refinement method
2637 [R(int) = 0.0226]
98.0%
Full-matrix
least-squares on F2
Data/restraints/parameters 2637/0/183
Goodness-of-fit on F2
1.000
= 0.0472, wR
Final R indices [I >2 (I)]
R
=
=
1
2
0.0830
= 0.1120, wR
R indices (all data)
R
1
2
0.1295
Largest diff. peak and hole
0.223 and -0.197 e Å-3
19

Table 2. Calculation results of molecular properties of the newly synthesized
compounds (3-6c) calculated using DFT at the B3LYP/6-31G (d, p) basis set in
gaseous phase.
(a.
E
(
E
(
LUMO
∆E(e
V)
IP(e
V)
EA(e
V)
(e
V)
η(eV
(e
V)
ω(eV
α
u)
μ(Deb
ye)
HOMO
eV)
eV)
)
)
3
4
5
-6.86 -1.49 5.36 1.980
19 64 55
-6.97 -2.14 4.82 1.548
16 92 24
-6.85 -2.48 4.37 2.368
67 47 21
-6.85 -2.54 4.30 2.361
51 59 92
-6.83 -2.52 4.31 2.537
77 28 49
-6.82 -2.51 4.31 2.585
90 71 19
8.62 -0.12 4.25 4.37 0.22 2.06 152.
72 66 03 69 85 37 23
8.72 0.38 4.55 4.16 0.24 2.48 158.
03 90 46 57 01 99 29
8.64 0.67 4.65 3.98 0.25 2.72 165.
67 06 87 80 08 10 06
8.61 0.80 4.70 3.90 0.25 2.84 169.
37 59 98 39 62 10 19
8.58 0.80 4.69 3.88 0.25 2.83 181.
28 79 53 74 72 56 65
8.56 0.81 4.69 3.87 0.25 2.83 194.
67 55 11 56 80 91 06
3
1
9
6
a
6
b
6
c
2
9
3
Table 3. Antimicrobial activity results (MIC) of the newly synthesized compounds
and the standard compounds.
MIC (ꢀg/mL)
Microorganism 3
Gram
4
5
6a
6b
6c
MMF
MP
negative
bacteria
S. choleraesuis >200
subsp.
200
>200
50
200
>200
600
>800
choleraesuis
Gram positive
bacteria
B. subtilis
200
150
100
50
75
100
800
600
20

L. lactis subsp. 150
lactis
100
75
50
75
100
600
>800
Yeasts
C. albicans
S. cerevisiae
Molds
75
50
50
25
25
25
50
25
75
>800
>800
>800
600
150
100
37.5
A. flavus
A. niger
P.
200
150
200
150
100
150
100
100
100
37.5
75
100
100
75
150
200
100
>800
>800
>800
600
600
600
37.5
chrysogenum
21

Figure(s)
Scheme 1
20

Figure(s)
Fig. 1 Crystal structure (Left) and packing diagram (Right) of compound 6b.
21

Figure(s)
-5.576e-2
5.576e-2
O5
O2
C4
O2
C2
C3
C4
C2
C3
C10
C11
C2
O3
O2
C3
O3
C7
C6
O5
C9
O4
C9
O3
C4
O5
C9
O4
O4
<O2-C2-C3-C4 6.25°
<C3-C4-O3-C9 -83.24° O4=-0. 553
<O3-C9-C10-C11 79.26° O5=-0. 602
O2=-0. 602
<O2-C2-C3-C4 -1.17° O2=-0. 596
<C3-C4-O3-C9 -73.37° O4=-0. 570
O5=-0. 592
<O2-C2-C3-C4 0.01°
<C6-C7-O3-C9 -38.76° O4=-0. 577
O2=-0. 598
O5=-0. 590
3
4
5
O5
C4
C3
O5
O2
C2
O3
O5
C14
C4
C3
O2
C2
C6
C7
C4
C3
O2
C2
O3
O3
C6
C7
C6
C7
C9
O4
C12
C13
C9
C9
O4
O6
O4
<O2-C2-C3-C4 -0.275°
<C7-C6-O3-C9 27.48°
<C12-O6-C13-C14 85.26° O5=-0. 596
O2=-0. 602
O4=-0. 576
<O2-C2-C3-C4 -0.24°
<C7-C6-O3-C9 24.56°
<C12-O6-C13-C14 86.14°
O2=-0. 602
O4=-0. 577
O5=-0. 596
<O2-C2-C3-C4 -0.297° O2=-0. 602
O4=-0. 575
<C7-C6-O3-C9 30.17°
O5=-0. 590
6c
6b
6a
Fig. 2 Selected dihedral angles, NBO charges and molecular electrostatic potential
maps of the newly synthesized compounds.
22

Figure(s)
5
3
4
6b
6a
6c
Fig. 3 HOMO and LUMO of compounds 3-6c.
23

Figure(s)
Fig. 4 Time-kill curves of compound 6a against S. choleraesuis subsp. choleraesuis
(A), L. lactis subsp. lactis (B) and S. cerevisiae (C).
24